肝癌相关成纤维细胞关键基因群的筛选、功能鉴定及干预研究

Tumor-associated fibroblasts (TAF) are the most predominant host-derived cells in the tumor microenvironment. Currently, whether there are intrinsic genetic and epigenetic changes in TAF remains elusive and considerable conflict results existed. Hepatocellular carcinoma (HCC), with the background of virus-related hepatitis and subsequent liver fibrosis or cirrhosis, is well-recognized as a typical microenvironment-related disease. In particular, the fundamental pathologic feature of liver fibrosis, that is the activation of hepatic satellite cells or portal fibroblasts, may serve as the most reach origin for TAF in hepatocarcinogenesis. In preliminary experiment, we performed whole-exome sequencing on TAF isolated from fresh tumor tissues of six HCC patients. Our results indicated that, although the genetic instability of TAF was far from that of the cancer cells, there were indeed a number of gene mutations in TAF. We also demonstrated that certain mutated genes greatly contributed to the tumor-promoting phenotype of TAF. Based on the above information, we hypothesized that genetic (i.e., gene mutations) and epigenetic (i.e., DNA methylation) changes should be responsible for the development and maintenance of tumor-promoting phenotype of TAF in HCC. By using whole-exomes sequencing and methylation array, we are aimed to identify the comprehensive molecular network and key pathways in TAF. Then, we will try to make clear how the genetically and epigenetically changed TAF promote the malignant transformation and disease progression of HCC, by focusing on their impact on the stemness of cancer cells, reprogramming inflammatory microenvironment and facilitating drug resistance. Finally, we will test the effectiveness and feasibility of anti-HCC treatments which targeting these genetically and epigenetically changed TAF.

肿瘤相关成纤维细胞(TAF)是肿瘤微环境最主要的宿主细胞，TAF是否存在内在的遗传学和表观遗传学异常尚无定论。发生在肝炎、纤维化/肝硬化背景的肝细胞癌是个典型的"微环境"相关肿瘤，肝纤维化的病理改变使得肝癌微环境TAF有了最广泛的来源。预实验中，我们对6例肝癌TAF的全外显子组测序的结果表明：尽管TAF 的遗传不稳定性远不及肝癌细胞，TAF的确存在一定数目的基因突变，某些基因的变异直接导致其促癌表型。本研究将利用先进的全外显子组测序技术，结合全基因组水平的甲基化检测，以"遗传（基因突变）"和"表观遗传(甲基化)"异常是肝癌TAF促癌表型形成和维持的内在因素为研究假设，制作超广谱的肝癌微环境TAF 的分子网络图谱；基于体内外微环境研究平台，拟阐明："基因突变型和甲基化异常型"TAF可通过影响癌细胞"干性"、调控免疫炎症微环境、介导耐药等机制促进肝癌发生发展和侵袭转移，是有效的抗肝癌治疗靶点。

肿瘤相关成纤维细胞(TAF)是肿瘤微环境最主要的宿主细胞，TAF是否存在内在的遗传学异常尚无定论。发生在肝炎、纤维化/肝硬化背景的肝细胞癌是个典型的"微环境"相关肿瘤，肝纤维化的病理改变使得肝癌微环境TAF有了最广泛的来源。在本项目中，通过对肝癌 TAF 的外显子组测序和表达谱检测、分析和验证，以及对TAF 促进肝癌发生发展和侵袭转移的机制研究，及作为治疗靶点的研究。系统阐明了TAF在肝癌发生发展中的重要作用、潜在临床意义和治疗价值。本项目（1）建立了酶消化法联合免疫磁珠分选进行原代TAF的分离、纯化平台，可获取高纯度的TAF。（2）有力的证实了，与正常成纤维细胞相比较，TAF确实存在大量的基因突变，但是这些突变可能并不是驱动TAF功能变化的主要因素，这些突变可能都是在微环境不利因素下的随机突变。（3）发现TAF具有促进肝癌进展的作用，其主要机制在于促进癌细胞的“干性”和EMT；筛选鉴定了一批具有同时抑制癌细胞和TAF的小分子化合物，具有潜在的治疗价值。（4）发现肝癌TAF较癌旁成纤维细胞的端粒酶显著缩短，是肝癌术后复发转移和生存的独立预测指标，具有重要的临床意义。共发表“标注本项目资助”的论文15篇（项目负责人高强为第一或通讯作者），其中SCI论文14篇（包括Gastroenterology、Hepatology、J Pathol等知名杂志）、中文核心期刊1篇。申请并获得国家发明专利2项（项目负责人高强均排第三位，专利号：ZL2015100031589，ZL2015100031837）。项目负责人在项目资助期间获得了：2015万人计划“青年拔尖人才”、2016国家优秀青年科学基金、2016教育部“青年长江学者”、2017上海市卫计委优秀学科带头人、2017上海市教委“曙光学者”等人才计划的支持。在本项目和其他项目的联合资助下，共培养1名硕士生和4名博士生。